Despite the enormous potential for human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) for development of new treatments for human disease, there still remain important gaps in our knowledge about the molecular mechanisms regulating establishment and maintenance of the pluripotent state. Improved understanding of fundamental mechanisms regulating pluripotency could improve the ability to establish pluripotent stem cells, in understanding how to maintain them in the undifferentiated state and how to differentiate them into specific cell lineages. The research proposed here seeks to provide a fundamentally better understanding of pluripotency and how it is controlled in hES cells and closely related iPCs. Maintenance of stem cells is known to be controlled by a group of core proteins that keep them in an undifferentiated state. When these proteins are downregulated they undergo differentiation into specialized cell types. Little is known about how the master regulatory circuitry is regulated other than feedforward positive interaction between the three core regulatory factors. Here we propose to study another protein that interacts with these core regulatory proteins and that may be a key regulator of their activity. These studies can expand our definition of the core stem cell regulatory circuitry. Through the research proposed here we will obtain a better understanding of the molecular processes at work when pluripotent ES cells decide to commit to lineage specific differentiation. For example, what genes must be turned off or on to achieve differentiation into specific lineages? How do chromatin modifications contribute to this regulation? This could lead to improvements in culture of hES cells and in methods for making iPSCs. A better understanding of these features could help better control these cells for use in regenerative medicine. Because hES cells are derived from the human embryo, these studies will also contribute important insights into human embryonic pre-implantation development.

Statement of Benefit to California:

A primary goal of Proposition 71 is to translate basic stem cell research to clinical applications. The disability and loss of earning power and personal freedom resulting from a disease or disorder are devastating and create a financial burden for California in addition to the suffering caused to patients and their families. Therapies using human embryonic stem cells (hESCs) and the related induced pluripotent stem cells (hiPSCs) have the potential to change millions of lives. Using hESCs and hiPSCs as models of disease will help us understand the underlying causes of disease and likely aid in the development of drugs to treat those diseases. However, for the potential of these cells to be realized, we need a better understanding of how they can be grown and what factors regulate the growth and self-renewal of the stem cell population. Maintenance of stem cells in an undifferentiated state is still problematical and long term growth of stem cells can be associated with appearance of genetic alterations some of which have previously been associated with cancer development. Moreover, understanding the mechanisms regulating stem cell growth will be important not only in maintaining stem cells but also in understanding how to drive their differentiation into more specialized cells. Finally, understanding the factors that support stem cell growth will be important for understanding the risks of transplanting stem cells and their differentiated derivatives into patients. Therefore, the raison d’etre for the proposed research is to provide a fundamentally better understanding of how hESCs and hiPSCs grow and self-renew. Anticipated benefits of our research to the Citizens of California include:
1. Development of improved methods for growing pluripotent stem cells and developing new cell-based treatments for a variety of diseases and disorders.
2. Development of improved understanding the risks of transplantation of stem cell-derived cells into patients and therefore improving the safety of stem cell-based transplantation.
4. Improved methods for understanding normal development of the early embryo
6. Transfer of new technologies and intellectual property to the public realm with resulting IP revenues coming into the state
7. Creation of new biotechnology spin-off companies based on generated intellectual property
8. Creation of new jobs in the biotechnology sector.
It is anticipated that, in the long term, the return to the State in terms of revenue, health benefits for its Citizens and job creation will be significant.

Progress Report:

Year 1

The goal of the project is to define the role of key regulators of pluripotency and differentiation in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). Work completed in the first reporting period of this project focused on a regulator named TCF-3. The role of this factor is known in mouse embryonic stem cell cultures to be a key regulator that helps define the pluripotent state in a very important way: loss of TCF-3 protein results in a refractory state of pluripotency and inability to differentiate. The molecular and cellular characteristics of TCF-3 and its function in human ESCs and iPSCs is not known. Therefore, it is not known whether human TCF-3 performs functions in hESC and hiPSC that are similar to its functions in mouse ESC. The work performed in the first funding period focused on defining the basic properties of human TCF-3 mRNA and protein expression under different culture conditions – preparatory experiments that will enable us to define its functions. Multiple conditions that enable maintenance of an embryonic stem cell state, or conditions that promote differentiation into different cell fate lineages were established. Biological tools were developed to study TCF-3 mRNA and protein under those conditions, including tools to re-introduce mutant and protein-modified TCF-3 and reagents to remove TCF-3 mRNA and protein from hESC cells. The following results were obtained: i) TCF 3 is expressed and active as a negative regulator, ii) TCF-3 protein is modified by protein cleavage and covalent modifications, iii) TCF-3 is an important regulator of differentiation genes. These data suggest that TCF-3 is an important modulator of stem cells and differentiation. While this might imply that TCF-3 performs similar functions in mouse and human ESC, we observe differences in the genes regulated and in hESC colony phenotypes when TCF-3 levels are modified. These observations suggest there are important differences in TCF-3 function that distinguish it from its counterpart in mouse ESC. Experiments have been designed to test this hypothesis and to define how the apparent differences of human TCF-3 are established at the molecular and cell signaling level.

Year 2

The goal of the project is to define the role of key regulators of pluripotency and differentiation in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). Specifically, this project focused on the regulation and actions of TCF-3. The role of this factor in mouse embryonic stem cell cultures is known to protect the pluripotent state and cellular responsiveness in a very important way: loss of TCF-3 protein results in a permanent state of pluripotency and inability to differentiate. However, the molecular and cellular characteristics of TCF-3 and its function in hESCs and iPSCs is not known, whether its functions are similar to its functions in mouse ESC. The work performed in the second funding period focused on mapping key protein modifications of TCF-3 including phosphorylation. Phosphorylation is a frequently used method to modify the actions of proteins – to either inhibit protein activity, stimulate activity, or change the ability to interact with other proteins. Human embryonic stem cell lines were modified to express a tagged version of TCF-3 for rapid isolation and purification from cell extracts. Purification was performed rapidly and under specialized conditions to prevent loss of any protein modifications. Mass spectrometry identified multiple phosphorylation sites and other forms of protein modification.
Work during this period also focused on developing strategies to identify key target genes regulated by TCF-3. Methods to remove TCF-3 from hESCs were developed and validated on levels to ensure bona fide removal without artefactual effects on hESCs. Removal of TCF-3, or “knockdown” will be used to investigate how hESCs cope in the absence of TCF-3. Preliminary results suggest that TCF-3 exerts control over a set of gene regulators as well as cellular signals. For these experiments, only early, immediate events that occur upon TCF-3 removal were assessed. A focus on early events is a purposeful effort to identify immediate-reacting, key regulatory steps. The results suggest that TCF-3 expression is dynamic, constitutively required, and that much of its function is to provide repressive actions. While this has general similarity to the role ascribed to TCF-3 in mouse embryonic stem cells, it appears that the types of genes and processes repressed by TCF-3 are distinct in hESC.

Year 3

The goal of the project is to define the role of key regulators of pluripotency and differentiation in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). Specifically, this project focused on the regulation and actions of TCF-3. The role of this factor in mouse embryonic stem cell cultures is known to protect the pluripotent state and cellular responsiveness in a very important way: loss of TCF-3 protein results in a permanent state of pluripotency and inability to differentiate. However, the molecular and cellular characteristics of TCF-3 and its function in hESCs is not known. It is not known whether TCF-3 functions are similar to its functions in mouse ESC. The work performed in the third funding period focused on defining the genes that are modulated by TCF-3 expression. Genome-wide microarray analysis was performed in the presence and absence of TCF-3 and in the presence and absence of differentiation conditions.
Removal of TCF-3, or “knockdown” revealed a specific, small set of genes that are dynamically dependent on TCF-3 for expression. Signals for differentiation that mimic the environment in the developing embryo were used to understand how TCF-3 functions in this setting. Preliminary data suggest that TCF-3 exerts a strong, noticeable function within the new signaling context. Additional microarray analyses, validation and biochemical confirmation are being used to complement the microarray studies and better define the precise role of TCF-3 action. The results suggest that TCF-3 expression is dynamic, constitutively required, and that much of its function is to provide repressive actions. While this has general similarity to the role ascribed to TCF-3 in mouse embryonic stem cells, it appears that the types of genes and processes repressed by TCF-3 are distinct in hESC.

Year 4

The goal of the project is to define the role of key regulators of pluripotency and differentiation in human embryonic stem cells (hESCs). Specifically, this project focused on the regulation and actions of the gene expression regulator TCF-3. The role of this factor in mouse embryonic stem cell cultures is known to protect the pluripotent state and cellular responsiveness in a very important way: loss of TCF-3 protein results in a permanent state of pluripotency and inability to differentiate. However, the molecular and cellular characteristics of TCF-3 and its function in hESCs is not known. It is not known whether TCF-3 functions are similar to its functions in mouse ESC. The work performed in the third funding period focused on defining the genes that are modulated by TCF-3 expression. Genome-wide microarray analysis was performed in the presence and absence of TCF-3 and in the presence and absence of differentiation conditions.
Removal of TCF-3, or “knockdown” revealed a specific, small set of genes that are dynamically dependent on TCF-3 for expression. Signals for differentiation that mimic the environment in the developing embryo were used to understand how TCF-3 functions in this setting. Our data suggests that TCF-3 exerts a strong, repressive function within the new signaling context. Microarray analyses, validation and biochemical confirmation have been used to better define the precise role of TCF-3 action. This past year utilized ChIP-seq approached to precisely define the pattern of genome occupancy that TCF-3 has in hESC cultures prior to differentiation. The results suggest that TCF-3 expression is dynamic, constitutively required, and that much of its function is to provide repressive actions. While this has general similarity to the role ascribed to TCF-3 in mouse embryonic stem cells, it appears that the types of genes and processes repressed by TCF-3 are distinct in hESC.